Gene therapy uses nucleic acids as treatment for genetic deficiencies and a large variety of acquired diseases and includes large DNA molecules (plasmid DNA; pDNA) but also small DNA (oligonucleotides; ODN) and RNA (ribozymes, SiRNA and mRNA) molecules. The success of gene therapy is largely dependent on the development of the gene delivery vector, which can be a viral vector or nonviral vector, such as a chemical carrier or delivery of naked DNA by physical methods. Nonviral vectors have many advantages over viral ones, including simple large-scale production, lack of immunogenicity, and low toxicity.
Cationic lipids capable of forming positively-charged liposomes are one of the most widely used nonviral vectors for gene delivery (Zhi et al., Bioconjugate Chemistry, 2013, 24: 478-519). Cationic lipids are amphiphilic molecules and generally consist of a hydrophobic domain (e.g., aliphatic chains, steroid rings), a hydrophilic headgroup (e.g., amines, quaternary ammonium salts, guanidiniums, heterocycles), and a linker group (e.g., ether, ester, carbamate or amide bond) connecting the two domains. The hydrophilic headgroup enables the condensation of nucleic acids by electrostatic interactions with the negatively-charged phosphate groups of the genes, and further governs transfection efficiency. Cationic lipids are usually formulated as cationic liposomes with a neutral co-lipid like dioleoyl phosphatidyl ethanolamine (DOPE) or cholesterol to improve transfection. When mixed with negatively-charged DNA, the positively-charged liposomes spontaneously form uniquely compacted structures called lipoplexes.
Solodin and co-workers reported the utilization of imidazolinium cationic lipids as synthetic carriers to deliver genes into cells (Solodin et al., Biochemistry, 1995, 34(41): 13537-13544). These lipids included 1-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)-heptadecenyl)-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), and its analogues 1-[2-(hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imidazolinium chloride (DPTIM) and 1-[2-(tetradecanoyloxy)ethyl]-2-tridecyl-3-(2-hydroxyethyl)-imidazolinium chloride (DMTIM). DOTIM was found to be the most effective among the three compounds for both in vitro transfection and for in vivo gene delivery. The structures of DMTIM, DPTIM, and DOTIM are as follows:

Methods for the preparation of aliphatic imidazolinium compounds starting with the multifunctional compound N,N′-bis(2-hydroxyethyl)ethylenediamine have been described in U.S. Pat. No. 5,705,655 (Heath), U.S. Pat. No. 5,830,878 (Gorman), and U.S. Pat. No. 8,044,215 (Yu). However, these prior processes exhibit various disadvantages. For example, in order to acylate the primary hydroxyl groups without concomitant acylation of the more nucleophilic secondary amines, the latter are protected with tert-butyloxycarbonyl groups. These protecting groups are commonly and herein referred to as “BOC” groups. This step requires the reagent di-tert-butyl dicarbonate, which is an expensive and toxic compound. Also, the BOC protecting groups have to be removed by acid hydrolysis in a subsequent step, which results in an additional amount of organic and aqueous waste.
Further, the acylation procedures of this BOC protected intermediate require the use of acid halides in the presence of base (e.g., triethylamine), or reaction with a carboxylic acid in the presence of N,N′-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP). The need for triethylamine or DMAP in the acylation reactions results in additional costs and waste. Further, the DCC/DMAP procedure results in formation of dicyclohexylurea (DCU) as a side product, and usually requires purification of the formed ester that may be labor-intensive.
There exists a need, therefore, for improved methods for preparing and purifying DOTIM and other amphiphilic imidazolinium compounds. In particular, there exists a need for such processes that are readily scalable, cost-effective, environmentally friendly, and capable of consistently yielding highly pure compounds.